Numerous industrial and agricultural processes e.g. municipality operations, food and feed processing and forestry generate biomasses, waste and by-products containing polymeric sugars e.g. in the form of starch, cellulose and hemicellulose. Agribusiness and chemical industries as well as public organisations have considerable interest in developing processes for converting such biomasses into materials of a higher value. Thus, by way of example such biomasses could potentially be converted into bio-ethanol, biogas or chemicals using microorganisms and/or hydrolytic enzymes. However, the majority of processes known today have not yet reached large-scale commercial practice due to their high production cost and high energy demand and thus inherent uncertain economic feasibility.
Besides being important as food and feed, carbohydrates from biomass can be used as feedstock for a number of industrial processes. In the form of polymers a well-known product is paper where cellulose is the main component. However, when processed into oligomers and monomers carbohydrates are an important feedstock for a number of industrial processes. As will be described in detail they are necessary for a number of microbial processes, but in addition they can be used as feedstock for e.g. enzymatic processing into specialty carbohydrates for food and feed e.g. trehalose. Also carbohydrate oligomers and monomers may substitute petrochemicals for processing into plastics and organic chemicals. Furthermore, carbohydrates may be used as hydrogen carriers in catalytic hydrogenation.
It is therefore evident that if a low-cost and abundant resource of processed carbohydrates can be made available for industrial processing it may have a substantial economic potential.
Starch is the most widespread storage carbohydrate in plants and occurs in the form of granules, which differ markedly in size and physical characteristics from species to species. Starch granules are generally quite resistant to penetration by both water and hydrolytic enzymes due to the formation of hydrogen bonds within the same molecule and with other neighbouring molecules. However, these inter- and intra-hydrogen bonds can become weak as the temperature of the suspension is raised. When an aqueous suspension of starch is heated, the hydrogen bonds weaken, water is absorbed, and the starch granules swell. This process is commonly called gelatinization because the solution formed has a gelatinous, highly viscous consistency. Chemically, starch is a natural polymer of glucose, which is generally insoluble but dispersible in water at room temperature and made up of a repeating unit similar to that of cellulose and linked together by α-1,4 and α-1,6 glucosidic bonds, as opposed to the β-1,4 glucosidic bonds for cellulose. The units form either a linear chain component, called amylose, or a branched chain component, called amylopectin. Most plant seeds, grains and tubers contain about 20-25% amylose. But some, like e.g. pea starch have 60% amylose and certain species of corn have 80% amylose. Waxy varieties of grains, such as rice, are low in amylose.
Apart from starch the three major constituents in plant biomass are cellulose, hemicellulose and lignin, which are commonly referred to by the generic term lignocellulose. Polysaccharide containing biomasses as a generic term include both starch and lignocellulosic biomasses.
Cellulose, hemicellulose and lignin are present in varying amounts in different plants and in the different parts of the plant and they are intimately associated to form the structural framework of the plant.
Cellulose is a homopolysaccharide composed entirely of D-glucose linked together by β-1,4-glucosidic bonds and with a degree of polymerisation up to 10,000. The linear structure of cellulose enables the formation of both intra- and intermolecular hydrogen bonds, which results in the aggregation of cellulose chains into micro fibrils. Regions within the micro fibrils with high order are termed crystalline and less ordered regions are termed amorphous. The micro fibrils assemble into fibrils, which then form the cellulose fibres. The partly crystalline structure of cellulose along with the microfibrillar arrangement, gives cellulose high tensile strength, it makes cellulose insoluble in most solvents, and it is partly responsible for the resistance of cellulose against microbial degradation, i.e. enzymatic hydrolysis.
Hemicellulose is a complex heterogeneous polysaccharide composed of a number of monomer residues: D-glucose, D-galactose, D-mannose, D-xylose, L-arabinose, D-glucuronic acid and 4-O-methyl-D-glucuronic acid. Hemicellulose has a degree of polymerisation below 200, has side chains and may be acetylated. In softwood like fir, pine and spruce, galactoglucomannan and arabino-4-O-methyl-glucuronoxylan are the major hemicellulose fractions. In hardwood like birch, poplar, aspen or oak, 4-O-acetyl-4-methyl-glucuronoxylan and glucomannan are the main constituents of hemicellulose. Grasses like rice, wheat, oat and switch grass have hemicellulose composed of mainly glucuronoarabinoxylan.
Lignin is a complex network formed by polymerisation of phenyl propane units and it constitutes the most abundant non-polysaccharide fraction in lignocellulose. The three monomers in lignin are p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, and they are most frequently joined through arylglyceryl-β-aryl ether bonds. Lignin is linked to hemicellulose and embeds the carbohydrates thereby offering protection against microbial and chemical degradation.
As stated above the processed biomasses could potentially be converted into bio-ethanol or chemicals using microorganisms and/or hydrolytic enzymes, or the carbohydrates from the processed biomasses could be used as feedstock for a number of industrial processes, e.g. enzymatic processing into specialty carbohydrates for food and feed or as substitutes for petrochemicals in the production of plastics and organic chemicals. In addition the processing of carbohydrates in biomass according to the present invention can be combined with separation and fractionation of the non-carbohydrate components. A particularly preferred use of a process according to the present invention is an integrated part of a process for bio-ethanol production.
Bio-ethanol production from polysaccharide containing biomasses can be divided into three steps: 1) pre-treatment, 2) hydrolysis of the polysaccharides into fermentable carbohydrates 3) and fermentation of the carbohydrates.
Pre-treatment is required if subsequent hydrolysis (e.g. enzymatic hydrolysis) of the polysaccharides requires the break down of an otherwise protecting structure (e.g. lignin) of the plant materials. Several pre-treatment techniques are known. For cereals and grains, this pre-treatment may be in the form of a simple dry milling in order to render the surfaces accessible, but for lignocellulosic biomasses thermal and/or chemical processes are needed as well. A polysaccharide containing biomass consisting of e.g. refined starch does not require said pre-treatment methods prior to enzymatic processing. Pre-treatment-processes may be based on acidic hydrolysis, steam explosion, oxidation, extraction with alkali or ethanol etc. A common feature of the pre-treatment techniques is that combined with the action of possible added reactants they take advantage of the softening and loosening of plant materials that occurs at temperatures above 100° C.
Following the pre-treatment, the next step in utilisation of polysaccharide containing biomasses for production of bio-ethanol or other biochemicals is hydrolysis of the liberated starch, cellulose and hemicellulose into fermentable sugars. If done enzymatically this requires a large number of different enzymes with different modes of action. The enzymes can be added externally or microorganisms growing on the biomass may provide them.
Cellulose is hydrolysed into glucose by the carbohydrolytic cellulases. The prevalent understanding of the cellulolytic system divides the cellulases into three classes; exo-1,4-β-D-glucanases or cellobiohydrolases (CBH) (EC 3.2.1.91), which cleave off cellobiose units from the ends of cellulose chains; endo-1,4-β-D-glucanases (EG) (EC 3.2.1.4), which hydrolyse internal β-1,4-glucosidic bonds randomly in the cellulose chain; 1,4-β-D-glucosidase (EC 3.2.1.21), which hydrolyses cellobiose to glucose and also cleaves of glucose units from cellooligosaccharides.
The different sugars in hemicellulose are liberated by the hemicellulases. The hemicellulytic system is more complex than the cellulolytic system due to the heterologous nature of hemicellulose. The system involves among others endo-1,4-β-D-xylanases (EC 3.2.1.8), which hydrolyse internal bonds in the xylan chain; 1,4-β-D-xylosidases (EC 3.2.1.37), which attack xylooligosaccharides from the non-reducing end and liberate xylose; endo-1,4-β-D-mannanases (EC 3.2.1.78), which cleave internal bonds; 1,4-β-D-mannosidases (EC 3.2.1.25), which cleave mannooligosaccharides to mannose. The side groups are removed by a number of enzymes; α-D-galactosidases (EC 3.2.1.22), α-L-arabinofuranosidases (EC 3.2.1.55), α-D-glucuronidases (EC 3.2.1.139), cinnamoyl esterases (EC 3.1.1.), acetyl xylan esterases (EC 3.1.1.6) and feruloyl esterases (EC 3.1.1.73).
The most important enzymes for use in starch hydrolysis are alpha-amylases (1,4-α-D-glucan glucanohydrolases, (EC 3.2.1.1). These are endo-acting hydrolases which cleave 1,4-α-D-glucosidic bonds and can bypass but cannot hydrolyse 1,6-alpha-D-glucosidic branchpoints. However, also exo-acting glycoamylases such as beta-amylase (EC 3.2.1.2) and pullulanase (EC 3.2.1.41) can be used for starch hydrolysis. The result of starch hydrolysis is primarily glucose, maltose, maltotriose, α-dextrin and varying amounts of oligosaccharides. When the starch-based hydrolysate is used for fermentation it can be advantageous to add proteolytic enzymes. Such enzymes may prevent flocculation of the microorganism and may generate amino acids available to the microorganism.
In combination with pre-treatment and enzymatic hydrolysis of lignocellulosic biomasses, it has been found that the use of oxidative enzymes can have a positive effect on the overall hydrolysis as well as the viability of the microorganisms employed for e.g. subsequent fermentation. The reason for this effect is the oxidative crosslinking of lignins and other phenolic inhibitors as caused by the oxidative enzymes. Typically laccase (EC 1.10.3.2) or peroxidase (EC 1.11.1.7) are employed either externally or by incorporation of a laccase gene in the applied microorganism.
Enzymatic hydrolysis of biomass has previously been described. However, in case of lignocellulosic biomasses only material consisting of fibres and particles with an average size below 1 inch (25.4 mm) and furthermore having a relatively low dry matter content, i.e. below 20% (w/w), have successfully been hydrolysed by such a method.
U.S. Pat. No. 4,409,329 describes hydrolysis of solid cellulose material to sugar, where cellulose is hydrolysed to simple sugars by treating a granular slurry of 3-20% (w/w) solid feed containing 30-80% (w/w) cellulose, with a cellulase enzyme complex. The solid cellulose-containing charge stock had a mean particle size from 0.01 to 1 inch (0.0254-25.4 mm) in diameter. Perforated rotorblades were used for mixing.
US2002117167A describes enzymatic hydrolysis of hemicellulose in biomass material, comprising solubilizing at least a portion of hemicellulose and hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. The utilised biomass is preferably aqueous slurry of raw or pre-treated material. The biomass material may be any cellulosic material that includes hemicellulose. The process is described as being especially effective with grain fibres such as corn, wheat, rice, oats or barley.
US2004005674A describes a process for enzymatic hydrolysis of lignocellulose. Degradation of lignocellulose to sugars comprises contacting the lignocellulose with at least one auxiliary enzyme and at least one cellulase. The lignocellulosic material was grounded (the average fibre size of the material was not further specified) and had a low dry matter content (0.2 g of grounded stover material in 10 ml of the enzyme solution).